The efficacy of many drugs has been linked to specific mutations or polymorphisms in genes. The era of personalized genetic medicine has become a reality. Today doctors are prescribing drugs based on genetic information. Infectious disease doctors are prescribing drugs for the treatment of AIDS based on resistance mutations found in the human immunodeficiency virus isolated from the patient (Rhee S Y, et al. Antimicrob Agents Chemother 2004; 48(8):3122-6). Polymorphisms in the cytochrome p450 gene are being used to determine a patient's drug-metabolizer status (Ingelman-Sundberg M. Trends Pharmacol Sci 2004; 25(4): 193-200). Mutations in the epidermal growth factor receptor gene activate a substantial clinical response to the drug gefitinib in 10% of non-small cell lung cancer patients (Lynch T J, et al. N Engl J Med 2004; 350(21): 2129-39).
Additionally, the early detection and treatment of cancer significantly reduces suffering and death. In fact, most cancers are curable when detected early. Molecular analysis of genomic DNA to detect a small number of cancerous cells in body fluids is a promising approach to the early detection of cancer.
Tumors transiently shed cells into adjacent body fluids. Malignant cells from localized tumors have been detected in blood, sputum, urine and stool. Sensitive detection of these rare cells is difficult because of the large excess of normal cells in these body fluids. The molecular signal generated from the normal or wildtype DNA from these cells overwhelms or out competes the minor mutant fraction signal.
The number of diseases that are linked to gene mutations continues to increase as the sequence of the human genome is unraveled. Many technologies have emerged to positively select for these minor mutations such that their signal can be amplified over the wildtype. These technologies can be divided into two types; those that detect known mutations and those that detect unknown mutations.
The sensitive detection of a mutation at a known site in DNA is readily done with existing technologies. Allele specific primers can be designed to target a mutation at a known location such that its signal can be preferentially amplified over wildtype DNA. Unfortunately, this is not possible with unknown mutations that may occur at any position (base) in the target sequence. Most mutations in cancer genes are not located at defined positions. The kras gene is highly mutated in cancer. Mutations are clustered in hotspots at exon 12 and 13, but can occur throughout the gene.
Nucleic acid sequencing is the ultimate standard for detecting nucleotide variations. Nucleic acid sequencing is also well suited for detecting unknown mutations or polymorphisms that may occur at any base within a target nucleic acid segment. The chemistry of enzymatic DNA sequencing, the most commonly used method, has essentially remained the same since its conception (Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977)). However, it has limitations related to cost, read length, and detection of mutations. The art has been improved by technology that has allowed for its automation such as the introduction of fluorescent dyes, robotics and improved electrophoretic systems with automated detection. However, if genetic variations occur at a low frequency in the sample population, automation comes at a cost that is too high for most laboratories. Even in a manual mode, sequencing can be cost prohibitive because it is labor intensive. This limits its widespread use as a detection tool, especially when the variation occurs at a low frequency in the sample population being tested. Moreover, the read length is limited by the resolution power of electrophoretic separation. The detection of mutations by DNA sequencing is not always obvious, especially if it compromises less than 20% of the wildtype sequence.
DNA microarrays, also known as gene chips and oligonucleotide microarrays, have been gaining more main stream use for detecting both known and unknown variations Hacia J G. Nat Genet. 1999; 21(1 Suppl):42-7). They are particularly suited for high-density analysis of a particular genetic sequence usually from a small number of individuals. But, because of high equipment and manufacturing costs, it is not as suited for large-scale population screening of unknown variations. Chips are also extremely inefficient at detecting insertions and deletion mutations. Increasing the complexity of chips by 2 to 4 fold by adding oligonucleotides to detect these mutations is problematic. Also, microarrays, like sequencing, do not have adequate sensitivity to detect mutations that may be present as a minor fraction in a vast amount of wildtype DNA. Thus, the discovery and detection of genetic variations linked to drug efficacy is hindered by the inadequacies of current technology. Therefore, there is a need in the art for a simple inexpensive process to screen nucleic acids for unknown nucleotide variations prior to sequencing.